Metabolic engineering for microbial production of terpenoid products

ABSTRACT

In various aspects and embodiments, the invention relates to bacterial strains and methods for making terpene and terpenoid products. The invention provides bacterial strains with improved carbon flux through the MEP pathway, to thereby increase terpene and/or terpenoid product yield by fermentation with carbon sources such as glucose.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalPatent Application No. 62/454,121, filed Feb. 3, 2017, the content ofwhich is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 2, 2018, isnamed MAN-008PC Sequence Listing.txt and is 20,480 bytes in size.

BACKGROUND

The food and beverage industries as well as other industries such as theperfume, cosmetic and health care industries routinely use terpenesand/or terpenoid products, including for use as flavors and fragrances.However, factors such as: (i) the availability and high price of theplant raw material; (ii) the relatively low terpene content in plant;and (iii) the tedious and inefficient extraction processes to producesufficient quantities of terpene products on an industrial scale allhave stimulated research on the biosynthesis of terpenes usingplant-independent systems. Consequently, effort has been expended indeveloping technologies to engineer microorganisms for convertingrenewable resources such as glucose into terpenoid products. Bycomparison with traditional methods, microorganisms have the advantageof fast growth without the need for land to sustain development.

There are two major biosynthetic routes for the essential isoprenoidprecursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate(DMAPP), the mevalonate (MVA) pathway and the methylerythritol phosphate(MEP) pathway. The MVA pathway is found in most eukaryotes, archaea anda few eubacteria. The MEP pathway is found in eubacteria, thechloroplasts of plants, cyanobacteria, algae and apicomplexan parasites.E. coli and other Gram-negative bacteria utilize the MEP to synthesizeIPP and DMAPP metabolic precursors. While the MEP pathway provides atheoretically better stoichiometric yield over the MVA pathway, the MEPpathway in E. coli and in other bacteria has a variety of intrinsicregulation mechanisms that control and/or limit carbon flux through thepathway. See, Zhao et al., Methylerythritol Phosphate Pathway ofIsoprenoid Biosynthesis, Annu Rev. Biochem. 2013; 82:497-530; Ajikumar PK, et al., Isoprenoid pathway optimization for Taxol precursoroverproduction in Escherichia coli. Science 2010; 330-70-74.

Microbial strains and methods for improving carbon flux through the MEPpathway are needed for industrial-scale production of terpenes andterpenoids in bacterial systems.

SUMMARY OF THE INVENTION

In various aspects, the invention relates to methods and bacterialstrains (such as E. coli) for making terpene and terpenoid products. Incertain aspects, the invention provides bacterial strains with improvedcarbon flux through the MEP pathway, to thereby increase terpene and/orterpenoid product yield by fermentation with carbon sources such asglucose. For example, in some embodiments the method comprises providinga bacterial strain that produces isopentenyl diphosphate (IPP) anddimethylallyl diphosphate (DMAPP) through the MEP pathway, and convertsthe IPP and DMAPP to a terpene or terpenoid product through a downstreamsynthesis pathway. The bacterial strain, when cultured with a carbonsource such as glucose, metabolizes greater than 1% of the carbonentering glycolysis through the MEP pathway, and in various embodimentsmetabolizes greater than 15% of the carbon entering glycolysis throughthe MEP pathway (greater than 15% “MEP carbon”).

In various embodiments, the invention involves “tuning” down expressionor activity of one or more competing enzymes or pathways in thebacterial production strain, such as the ubiquinone synthesis pathway,without substantial or measurable impact on strain growth or viability.In some embodiments, the expression or activity of the IspB enzyme isdecreased, optionally by modification to the ribosomal binding sequence(RBS), promoter, or amino acid sequence, or replacement with an IspBortholog.

Alternatively, or in addition, the invention involves increasing theavailability or activity of Fe—S cluster proteins, so as to supporthigher activity of the Fe—S enzymes IspG and/or IspH, optionally byaltering expression of the isc operon, and/or by deletion of the ryhBsmall RNA.

Alternatively, or in addition, in some embodiments the inventioninvolves tuning the activity of IspG and/or IspH by overexpressionand/or by selection of beneficial mutants or ortholog(s). Such mutantsor orthologs can increase MEP carbon by pulling carbon further down theMEP pathway. Alternatively, or in addition, MEP enzyme complementationas evaluated by metabolomics can identify MEP enzyme complementationthat results in high MEP carbon, with carbon pulled further down thepathway to the MEcPP intermediate.

In certain embodiments, the bacterial cell produces one or moreterpenoid compounds, such as monoterpenoids, sesquiterpenoids, andditerpenoids, among others. Such terpenoid compounds find use inperfumery (e.g., patchoulol), in the flavor industry (e.g., nootkatone),as sweeteners (e.g., steviol glycosides), or therapeutic agents (e.g.,taxol).

The host cell will generally contain a recombinant downstream pathwaythat produces the terpene or terpenoid from IPP and DMAPP precursors.

The recovered terpene or terpenoid may be incorporated into a product(e.g., a consumer or industrial product). For example, the product maybe a flavor product, a fragrance product, a sweetener, a cosmetic, acleaning product, a detergent or soap, or a pest control product. Thehigher yields produced in embodiments of the invention can providesignificant cost advantages as well as sustainability and qualitycontrol of the terpene or terpenoid ingredient.

In other aspects, the invention provides bacterial cells, such as E.coli, having one or more genetic modifications that increase MEP carbon,as described in detail herein.

Other aspects and embodiments of the invention will be apparent from thefollowing detailed description of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic for terpenoid production through the MEP pathway.A bacterial cell is represented, taking in glucose as a carbon source.Glucose is converted to biomass through the TCA cycle or funneledthrough the MEP pathway to the desired terpenoid products. Glucose comesinto the cell and is converted to pyruvate (PYR) withglyceraldehyde-3-phosphate as an intermediate (GAP). PYR and GAP arecombined to make DOXP, which is converted to MEP and commits the pathwayto FPP (going through MEcPP). DOX and ME are dephosphorylated productsof DOXP and MEP, respectively. DOX, ME, and MEcPP are found outside thecell. The more flux that is forced into the MEP pathway, the more theseproducts are found extracellularly. These side products can be used asmarkers of bottlenecks in the MEP pathway, and to identify targets forengineering. Black arrows show enzyme-mediated biochemical reactionstowards terpenoids, light grey arrows show a competing side product,dark grey arrows show transport of a product outside of the cell, andwhite arrows show condensed pathways for simplicity.

FIG. 2 illustrates genetic modifications to E. coli strain chassis toimprove terpenoid production via the MEP pathway for an exemplaryterpenoid product (Product A).

FIG. 3 shows the modifications to the Product A strain in going fromStrain G2 to Strain G4 (see FIG. 2). These modifications enable thecarbon to move ‘downstream’ in the MEP biochemical pathway, pushing theintermediate product pools from DOXP (and extracellular DOX) to MEP (andextracellular ME). The extracellular accumulation of large pools of MEPpathway intermediates can be used to inform engineering designsintending to push carbon flux down the pathway and on to MEcPP. FIG. 3shows experiments where a shift of 40% carbon from DOX to ME is observedin going from Strain G2 to G4.

FIG. 4 shows genetic modifications to E. coli strain chassis to improveterpenoid production via the MEP pathway for Product B. The pgi mutantmodification was identified in a high-production clone.

FIG. 5 shows results from a transcriptome profiling experiment providingevidence for the involvement of ryhB and the isc operon. The expressionof ryhB is upregulated, and the isc operon is generally down-regulated,in E. coli in response to the installation of a downstream terpenoidpathway for the production of Product A or Product B.

FIG. 6 shows the combined effect of ryhB and isc operon modifications.Given the importance of iron and iron-sulphur (Fe—S) clusterbiochemistry in MEP terpenoid metabolism, a series of modifications tothe production chassis was performed to increase titer of product(Product A). In sequential order, the G2 Product A chassis had the ryhBdeleted, then the wild-type promoter of iscS was replaced with aconstitutive promoter sequence, and the native iscR gene was deleted.With each modification, titer of product A increases.

FIG. 7A and FIG. B illustrate IspG enzyme engineering. Mutationlibraries of wild-type E. coli IspG gene were designed based onpredicted changes to a structural model (FIG. 7A). Libraries weredesigned for improved kinetics, protein stability, or strain robustness.Specific variants were introduced at discrete locations in the sequencein each separate library (FIG. 7B). Nine total combinatorial librarieswere designed using sequence alignments to ˜1000 diverse ispG orthologs.

FIG. 8 shows screening of the mutation libraries of wild-type E. coliispG gene, to replace the wild-type gene in three strains that makeProduct A. Preliminary screening of the integrated libraries resulted in20-40% of the introduced variants giving improved product titers, withup to 1.5× increase in terpenoid product observed.

FIG. 9 shows the results of validation and secondary re-screening ofleads resulting from the primary screen. The G11 variant wasincorporated into production chassis for both Products A and B.

FIG. 10 shows results upon supplementing the wild-type ispG gene in theproduction strains with the selected mutated version. Thesupplementation results in a 20% increase in product titer. The completetranslation of the improvement regardless of the terpenoid productsuggests the possibility of a ‘universal chassis’ that can support thehigh-level production of any terpenoid via the MEP pathway.

FIG. 11 shows tuning the translation rate of ispB via modification ofthe native RBS sequence, which improves the production of terpenoidproduct. Various mutants of the wild-type (WT) ispB RBS (ribosomebinding site) were introduced over the native ispB RBS to tune proteintranslation. These RBS changes impact terpenoid production, some of themresulting in improved production. Several hits were identified from theprimary screening, giving strains with improvement of 1.7× for Product Aand 2× for Product B. The left-most column is for the parental control,which sets the value of 1. Lead hits are repeated and validated, fullysequenced, and then transferred into the lead generation of each productstrain.

FIG. 12 shows four mutant strains validated through secondary screeningwith replication, confirming the improvement observed in the primaryscreen of ispB RBS.

FIG. 13 shows that overexpression of MEP genes causes drop in producttiter. Increasing the expression level of MEP genes over the G5 parentlevels results in ˜50% less terpenoid Product A produced, with strongerexpression (+++) exacerbating the decrease over weaker expression (+).In contrast, the addition of ispG and ispH genes to an operon expressingdxr enables recovery of product titer to parental levels, indicatingthat careful balancing of gene expression in this pathway will enablehigh terpenoid product titers.

FIG. 14 shows that, though titer drops, more carbon goes into MEPpathway. While the MEP pathway complementation work showed that ProductA titer dropped or stayed the same in response to gene expressionchanges, the intermediate MEP pathway metabolites increasedsignificantly more in concentration. MEP carbon metabolites werequantified via liquid chromatography and mass spectrometry againstauthentic standards, and found to significantly increase in complementedstrains compared to control G5 strains. The resulting metaboliteconcentrations are expressed in terms of molarity, to focus on the flowof carbon molecules through the MEP pathway to the desired product. Themajority of MEP intermediates are observed outside the cell, with DOX,ME, and MEcPP representing the majority; the major intracellular productis CDP-ME, with increasing accumulation observed in complementedstrains. While dxr overexpression caused the Product A titer to drop by2×, the amount of carbon entering the MEP pathway and accumulating asintermediates went up 6×; that is, more carbon is entering the MEPpathway, but less of it is getting out. Moreover, while the G5 parentaccumulated mostly DOX (and some MEcPP), the increase in dxr (whichconverts DOXP to MEP) shifts the carbon down the pathway, resulting inmore carbon pooling extracellulary as ME and MEcPP. Increasing ispEexpression on top of dxr further increase the amount of carbon in theMEP pathway to almost 10× over the G5 parent, and shifted almost all ofthat carbon downstream to MEcPP. Interestingly, overexpressing ispG andispH in addition to dxr gives you a very similar intermediate profile tooverexpressing dxr only, though the product titer is doubled in theformer instance.

FIG. 15 shows the total amount of Product A that would be produced ifall carbon accumulating in the MEP pathway in FIG. 14 was successfullyconverted through to final product. The data shows production potentialof the G5 strain complemented with an empty plasmid versus the sameplasmid backbone expressing MEP pathway genes (with varied expressionlevel increasing from + to +++). Converting the total amount of carbonin the MEP pathway (quantified as in FIG. 14) into Product A Equivalentsshows that the carbon flux through the pathway increases withcomplementation, with almost 9× more Product A potential made possiblein these strains.

DETAILED DESCRIPTION OF THE INVENTION

In various aspects, the invention relates to bacterial strains andmethods for making terpene and terpenoid products. In certain aspects,the invention provides bacterial strains with improved carbon fluxthrough the MEP pathway, to thereby increase terpene and/or terpenoidproduct yield by fermentation with carbon sources such as glucose. Forexample, in some embodiments the method comprises providing a bacterialstrain that produces isopentenyl diphosphate (IPP) and dimethylallyldiphosphate (DMAPP) through the MEP pathway, and converts the IPP andDMAPP to a terpene or terpenoid product through a downstream synthesispathway. The bacterial strain, when cultured with a carbon source suchas glucose, metabolizes greater than 1% of the carbon enteringglycolysis through the MEP pathway (greater than 1% “MEP carbon”). Insome embodiments, at least about 5%, at least about 10%, at least about15%, at least about 17%, or at least about 20% of carbon enteringglycolysis becomes MEP carbon. In still other embodiments, at leastabout 22% or at least about 25% or at least about 27% of carbon enteringglycolysis becomes MEP carbon. With glucose as carbon source, thetheoretical maximum for carbon entering the MEP pathway is about 30% inE. coli. In some embodiments, the strain substantially meets thistheoretical maximum yield of MEP carbon, meaning that the strainprovides at least about 80% of the maximum theoretical yield. Prioryields of MEP carbon reported in the literature are less than 1%. See,Zhou K, Zou R, Stephanopoulos G, Too H-P (2012) Metabolite ProfilingIdentified Methylerythritol Cyclodiphosphate Efflux as a Limiting Stepin Microbial Isoprenoid Production. PLoS ONE 7(11): e47513.doi:10.1371/journal.pone.0.0047513.

In various embodiments, the microbial strain is a bacteria selected fromEscherichia spp., Bacillus spp., Rhodobacter spp., Zymomonas spp., orPseudomonas spp. In some embodiments, the bacterial species is selectedfrom Escherichia coli, Bacillus subtilis, Rhodobacter capsulatus,Rhodobacter sphaeroides, Zymomonas mobilis, or Pseudomonas putida. Insome embodiments, the bacterial strain is E. coli.

The host bacterial cell expresses an MEP pathway producing isopentenyldiphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Glucose comesinto the cell and is converted to pyruvate (PYR) withglyceraldehyde-3-phosphate as an intermediate (G3P or GAP). G3P and PYRare combined to make 1-deoxy-D-xylulose-5-phosphate (DOXP), which isconverted to 2-C-methyl-D-erythritol 4-phosphate (MEP) and commits thepathway to IPP and DMAPP. DOX, ME, and MEcPP are found outside the cell.The more flux into the MEP pathway, the more these products are foundextracellularly. See FIG. 1.

The MEP (2-C-methyl-D-erythritol 4-phosphate) pathway is also called theMEP/DOXP (2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose5-phosphate) pathway or the non-mevalonate pathway or the mevalonicacid-independent pathway. The pathway typically involves action of thefollowing enzymes: 1-deoxy-D-xylulose-5-phosphate synthase (Dxs),1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr, or IspC),4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD),4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE),2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF),1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG),1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IspH) andisopentenyl diphosphate isomerase (Idi). The MEP pathway, and the genesand enzymes that make up the MEP pathway, are described in U.S. Pat. No.8,512,988, which is hereby incorporated by reference in its entirety.Thus, genes that make up the MEP pathway include dxs, dxr (or ispC),ispD, ispE, ispF, ispG, ispH, idi, and ispA.

IPP and DMAPP (the products of the MEP pathway) are the precursors ofterpenes and terpenoids, including monoterpenoids, sesquiterpenoids,diterpenoids, and triterpenoids, which have particular utility in theflavor, fragrance, cosmetics, and food sectors. Synthesis of terpenesand terpenoids proceeds via conversion of IPP and DMAPP precursors togeranyl diphosphate (GPP), farnesyl diphosphate (FPP), or geranylgeranyldiphosphate (GGPP), through the action of a prenyl transferase enzyme(e.g., GPPS, FPPS, or GGPPS). Such enzymes are known, and are describedfor example in U.S. Pat. No. 8,927,241, WO 2016/073740, and WO2016/029153, which are hereby incorporated by reference in theirentireties.

As used herein, the term “MEP carbon” refers to the total carbon presentas an input, intermediate, metabolite, or product of the MEP pathway.Metabolites include derivatives such as breakdown products, and productsof phosphorylation and dephosphorylation. MEP carbon includes productsand intermediates of downstream pathways including terpenoid synthesispathways. For purposes of this disclosure, MEP carbon includes thefollowing inputs, intermediates, and metabolites of the MEP pathway:D-glyceraldehyde 3-phosphate, pyruvate, 1-deoxy-D-xylulose-5-phosphate,1-deoxy-D-xylulose, 2-C-methyl-D-erythritol-5-phosphate,2-C-methyl-D-erythritol, 4-diphosphocytidyl-2-C-methyl-D-erythritol,2-phospho-4-diphosphocytidyl-2-C-methyl-D-erythritol,2C-methyl-D-erythritol 2,4-cyclodiphosphate,1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, isopentenyl diphosphate,and dimethylallyl diphosphate. MEP carbon further includes intermediatesand key metabolites in the downstream terpenoid synthesis pathwayexpressed by the cell. While the identity will vary based upon pathwayand enzymes employed, such products include: geranyl diphosphate (GPP),farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), orgeranylfarnesyl diphosphate (FGPP); their monophosphorylated versionsgeranyl phosphate, farnesyl phosphate, geranylgeranyl phosphate, orgeranylfarnesyl phosphate; their alcohols geraniol, farnesol,geranylgeraniol, or geranylfarnesol; as well as downstream terpene andterpenoid products. MEP carbon further includes compounds derived fromFPP or pathways that use FPP, including squalene, undecaprenyldiphosphate (UPP), undecaprenyl phosphate, octaprenyl diphosphate (OPP),4-hydroxybenzoate, 3-octaprenyl-4-hydroxybenzoate, 2-octaprenylphenol,3-octaprenylbenzene-1,2-diol,2-methoxy-6-octaprenyl-2-methoxy-1,4-benzoquinol,6-methoxy-3-methyloctaprenyl-1,4-benzoquinol, 3-demethyluibquinol-8,ubiquinol-8, ubiquinone, 2-carboxy-1,4-naphthoquinol,demethylmenaquinol-8, menaquinol-8, and menaquinone. MEP carbon furtherincludes isoprenol, prenol, isopentenyl phosphate, and dimethylallylphosphate metabolites. MEP carbon further includes prenylatedmetabolites and proteins, including prenylated indole. MEP carbon (theintermediates and metabolites above) can be quantified by massspectrometry (MS), such as tandem mass spectrometry (MS/MS) via triplequadrupole (QQQ) mass detector. An exemplary system is Agilent 6460 QQQ;alternatively with quantitative time-of-flight (QTOF), time-of-flight(TOF), or ion trap mass detectors.

Exemplary terpene or terpenoid products that may be produced inaccordance with the invention are described in U.S. Pat. No. 8,927,241,which is hereby incorporated by reference, and include: alpha-guaiene,alpha-sinensal, amorphadiene, artemisinic acid, beta-bisabolene,beta-Thujone, Camphor, Carveol, Carvone, Cineole, Citral, Citronellal,Cubebol, Geraniol, Limonene, Menthol, Menthone, Myrcene, Nootkatone,Nootkatol, Patchouli, Piperitone, Rotundone, Rose oxide, Sabinene,Steviol, Steviol glycoside (including Rebaudioside D or Rebaudioside M),Taxadiene, Thymol, and Valencene. Enzymes for recombinantly constructingthe pathways in E. coli are described in U.S. Pat. No. 8,927,241, WO2016/073740, and WO 2016/029153, which are hereby incorporated byreference.

In some embodiments, the microbial strain has at least one additionalcopy of one or more of dxs, ispD, ispF, and/or idi genes, which can berate limiting, and which can be expressed from an operon or module,either on a plasmid or integrated into the bacterial chromosome. In someembodiments, the bacterial strain has at least one additional copy ofdxs and idi expressed as an operon/module; or dxs, ispD, ispF, and idiexpressed as an operon or module. In these embodiments, the strainprovides increased flux through the MEP pathway as compared to wildtype. Complementation of the MEP pathway as described below, can be inaddition to dxs, ispD, ispF, and/or idi overexpression.

In various embodiments, the invention involves “tuning” down expressionor activity of one or more competing enzymes or pathways, such as theubiquinone synthesis pathway or the IspB enzyme, which competes for FPP.Alternatively, or in addition, the invention involves increasing theavailability or activity of Fe—S cluster proteins, so as to supporthigher activity of IspG and IspH, which are Fe—S enzymes. Alternatively,or in addition, in some embodiments the invention involvesoverexpression or tuning the expression or activity IspG and/or IspH,for example by selection of beneficial mutants or orthologs that havethe effect of increasing MEP carbon by pulling carbon further down thepathway. Alternatively, or in addition, further MEP enzymecomplementation as evaluated by metabolomics can identify MEP enzymecomplementation that results in high MEP carbon. These and otherembodiments are described in detail below.

In various embodiments, the microbial strain provides substantialincreases in MEP carbon, without substantial impact on strain growth andviability with glucose as a carbon source, for example, as determined byoptical density (O.D.) in culture, peak O.D., and/or growth rate. Forexample, despite modifications to one or more essential genes orpathways as described herein, the microbial strain does not exhibit adrop in peak O.D. of more than about 20%, or in some embodiments, doesnot exhibit a drop in peak O.D. of more than about 15%, or more thanabout 10%, or more than about 5%. In some embodiments, the strain doesnot exhibit a measurable impact on strain growth or viability, asdetermined for example by measuring growth rate or peak O.D.

In some embodiments, the ubiquinone biosynthesis pathway isdownregulated, for example, by reducing the expression or activity ofIspB, which uses IPP and FPP substrate. The ispB gene encodes anoctaprenyl diphosphate synthase that controls the synthesis ofubiquinone and thus directly competes with FPP synthase for IPP andDMAPP precursors. IspB is an essential gene in E. coli. See, Kainou T,et al., Dimer formation of octaprenyl-diphosphate Synthase (IspB) isessential for chain length determination of ubiquinone, J. Biol. Chem.276(11):7867-7883 (2001). However, decreasing the amount of IspB enzymein the cell by modifying the ispB RBS sequence and turning downtranslation of the mRNA can shift carbon flux towards the terpenoidrecombinant pathway, without substantial or measurable impact on thegrowth and viability of the strain. In some embodiments, IspB activityor expression is reduced to about 80% or less of the parent strain, orabout 70% or less of the parent strain, or 50% or less of the parentstrain, or 40% or less of the parent strain, or 25% or less of theparent strain.

The Shine-Dalgarno (SD) sequence is the ribosomal binding site inbacteria and is generally located around 8 bases upstream of the startcodon AUG. The RNA sequence helps recruit the ribosome to the messengerRNA (mRNA) to initiate protein synthesis by aligning the ribosome withthe start codon. In some embodiments, the six-base SD sequence in theispB gene is changed to CGTGCT or CGTGCC, or a modification thereofhaving one or two nucleotide changes from CGTGCT or CGTGCC. In suchembodiments, the IspB translation is tuned down, allowing for increasedcarbon flux to terpenoid biosynthesis, without substantial or measurableimpact on E. coli growth and viability.

In some embodiments, the expression or activity of IspB is modified byaltering expression of the ispB gene, that is, by modifying the promoterregion to decrease transcription, or by enhancing the rate ofdegradation of the ispB RNA or encoded protein. In some embodiments, theactivity of the ispB enzyme is decreased by mutation of the ispB aminoacid sequence or selection of an ispB ortholog with decreased activityin the bacterial strain. The wild type IspB amino acid sequence from E.coli is provided herein as SEQ ID NO:3. In some embodiments, from 1 toabout 10, or from 1 to about 5, amino acid substitutions, deletions,and/or insertions are made to the IspB amino acid sequence (SEQ ID NO:3)to decrease the activity of the protein, including one or moresubstitutions to the substrate binding site and/or active site. In someembodiments, the amino acid sequence (whether an ortholog or mutantsequence) has from about 50% to about 99% sequence identity with SEQ IDNO:3, or about 60% to about 99% sequence identity to SEQ ID NO:3, orfrom about 70% to about 99% sequence identity to SEQ ID NO:3, or fromabout 80% to about 99% sequence identity to SEQ ID NO:3, or from about90% to about 99% sequence identity to SEQ ID NO:3, or from about 95% toabout 99% sequence identity with the amino acid sequence of SEQ ID NO:3.Such mutants and orthologs can be informed by Kainou T, et al., Dimerformation of octaprenyl-diphosphate Synthase (IspB) is essential forchain length determination of ubiquinone, J. Biol. Chem.276(11):7867-7883 (2001); or Han, et al., Crystal structures ofligand-bound octaprenyl pyrophosphate synthase from Escherichia colireveal the catalytic and chain-length determining mechanisms Proteins2015 January; 83(1):37-45.

In some embodiments, the bacterial strain supports enhanced biology ofFe—S enzymes, such as IspG and/or IspH, to potentially improve theamount of MEP carbon in the strain. The IspG and IspH enzymes in the MEPpathway are iron-sulphur cluster enzymes, meaning that they need aspecial arrangement of Fe—S ions in their active sites to function anddo their chemistry. These Fe—S clusters are critical to life, and areincredibly sensitive to oxygen and oxygenation, which inactivates them.Under either anaerobic or aerobic growth conditions, iscR repressestranscription of the operon iscRSUA, which encodes genes for the Fe—Scluster biogenesis pathways. The repression of the isc operon by iscRresponds to the demand for the Fe—S cluster in the medium, because iscRhas to be bound to an Fe—S group to be able to repress the transcriptionof the isc operon. Under anaerobiosis, the demand for the Fe—S group islower than under aerobiosis, and therefore the repression of the operonis stronger than under aerobiosis. iscR recognizes and binds twoDNA-binding sites that overlap the promoter sequence to represstranscription of the iscRSUA operon. When the protein is bound to thesesites, the RNA polymerase is most likely not able to bind to thepromoter region to start transcription.

In some embodiments the isc operon is expressed under conditions usedfor terpene or terpenoid production using either a strong, intermediate,or weak bacterial promoter. The strength of the promoter can be variedand tuned to improve terpenoid product. The promoter can be constitutiveor inducible. In some embodiments, the promoter is a strong constitutivepromoter.

Upon deleting the promoter and first gene of the isc operon (namelyiscR) and replacing it with a constitutive or inducible promoter todrive expression of the genes remaining in the operon, improvements iniron-sulfur cluster enzyme performance are obtained. Thus, in someembodiments, the bacterial strain (e.g., E. coli) contains an iscRdeletion, with inducible or constitutive overexpression of iscSUA.Inducible and constitutive promoters for E. coli are known, and can beselected by one of skill in the art to tune expression of the operon. Invarious embodiments, the iscR gene is fully or partly deleted, or isinactivated by one or more mutations to the RBS or start codon. In someembodiments, the iscR gene is inactivated by amino acid mutation. In thevarious embodiments, the modifications to Fe—S enzyme regulationincrease MEP carbon without substantial or measurable impact on growthor viability of the strain, including under the aerobic or microaerobicconditions often used for terpenoid production in E. coli. The iscoperon is further reviewed in Santos JA, What a difference a clustermakes: The multifaceted roles of IscR in gene regulation and DNArecognition, Biochim. Biophys. Acta 1854(9):1102-12 (2015).

In some embodiments, the E. coli contains a ryhB deletion orinactivation. RyhB is a small RNA which acts to reduce iron consumptionunder low-iron conditions by downregulating expression ofiron-containing proteins, including enzymes of the TCA cycle and theaerobic respiratory chain. In addition, ryhB promotes synthesis of thesiderophore enterobactin. RyhB is a small RNA of approximately 90 nt inlength. RyhB promotes cleavage of the polycistronic iscRSUA mRNA betweenthe iscR and iscS open reading frames. The IscR-encoding 5′ fragmentremains stable, while the iscSUA 3′ fragment appears to be degraded. SeeMandin et al., (2016) A regulatory circuit composed of a transcriptionfactor, IscR, and a regulatory RNA, RyhB, controls Fe-S clusterdelivery, mBio 7(5):e00966-16. In various embodiments, deletion orinactivation (e.g., by nucleotide mutation) of ryhB increases MEPcarbon, without substantial or measurable impact on strain growth orviability, including under aerobic, microaerobic, or anaerobicconditions used for screening of strains or production of terpenoids.

In some embodiments, MEP enzyme expression or activity, is altered orbalanced to move carbon further down the pathway, for example, bycomplementation with additional enzyme copies, whether on plasmids orintegrated into the genome. As shown in FIG. 13, overexpression of MEPgenes can cause a drop in product titer. For example, increasing theexpression level of MEP genes over the G5 parent levels results in ˜50%less terpenoid Product A produced, with stronger expression (+++)exacerbating the decrease over weaker expression (+). In contrast, theaddition of ispG and ispH genes to an operon expressing dxr enablesrecovery of product titer to parental levels, indicating that carefulbalancing of gene expression in this pathway will enable high terpenoidproduct titers.

While Product A titer dropped or stayed the same with MEPcomplementation, the intermediate MEP pathway metabolites increasedsignificantly more in concentration. The majority of MEP intermediatesare observed outside the cell, with DOX, ME, and MEcPP representing themajority; the major intracellular product is CDP-ME, with increasingaccumulation observed in complemented strains. While dxr overexpressioncaused the Product A titer to drop by two-fold, the amount of carbonentering the MEP pathway and accumulating as intermediates went upsix-fold, that is, more carbon is entering the MEP pathway, but less ofit is getting out. Moreover, while the G5 parent accumulated mostly DOX(and some MEcPP), the increase in dxr (which converts DOXP to MEP)shifts the carbon down the pathway, resulting in more carbon poolingextracellulary as ME and MEcPP. Increasing ispE expression on top of dxrfurther increases the amount of carbon in the MEP pathway to almostten-fold over the G5 parent, and shifted almost all of that carbondownstream to MEcPP. Interestingly, overexpressing ispG and ispH inaddition to dxr gives a very similar intermediate profile tooverexpressing dxr only, though the product titer is doubled in theformer instance.

In some embodiments, the bacterial strain contains an overexpression ofdxr or homolog or ortholog thereof and ispG and/or ispH, optionally withone or more of an overexpression of ispD, ispE, and ispF. The genes canbe overexpressed individually or in an operon. The additional copies canbe expressed from a plasmid or integrated into the genome.Overexpression of dxr pushes DOXP to ME and MEcPP and pushes thebottleneck downstream. See FIG. 1. In some embodiments, dxr and ispE (orhomologs, orthologs, or derivatives thereof) are overexpressed, whichpushes DOXP to primarily MEcPP. See FIG. 14. Upon shifting thebottleneck to MEcPP, a tripling of MEP pathway potential can be observed(FIG. 15).

The wild type Dxr amino acid sequence from E. coli is provided as SEQ IDNO:4. In some embodiments, wild-type dxr activity is complemented withone or more additional gene copies, which may encode the wild-typeenzyme, or may encode a non-native or modified enzyme with one or moreamino acid modifications (e.g., from one to ten modificationsindependently selected from substitutions, insertions, and deletions) toincrease enzyme activity or stability. In some embodiments, thebacterial strain is complemented with a dxr ortholog having higheractivity than the E. coli enzyme.

In some embodiments, the bacterial strain expresses Brucella abortus DRLenzyme (SEQ ID NO: 8), which is a Dxr-like enzyme having low sequencehomology to E. coli Dxr. Perez-Gil, J., et al., 2012. Crystal structureof brucella abortus deoxyxylulose-5-phosphate reductoisomerase-like(DRL) enzyme involved in isoprenoid biosynthesis. Journal of BiologicalChemistry, 287(19), pp. 15803-15809. In some embodiments, the DRL enzymehas one or more amino acid modifications (e.g., from one to tenmodifications independently selected from substitutions, insertions, anddeletions) that improve the properties of the enzyme for activity and/orexpression in the bacterial host cell.

For example, in various embodiments the bacterial strain overexpressesor is complemented with a Dxr enzyme having 50% or more sequenceidentity with SEQ ID NO: 4 or 8, or at least about 60% sequenceidentity, or at least about 70% sequence identity, or at least about 80%sequence identity, or at least about 90% sequence identity, or at leastabout 95% sequence identity, or at least 97% sequence identity with theamino acid sequence of SEQ ID NO: 4 or 8. In some embodiments, the Dxrenzyme comprises from 1 to about 10, or from 1 to about 5, amino acidsubstitutions, deletions, and/or insertions with respect to the Dxr orDRL amino acid sequence (e.g., SEQ ID NO: 4 or 8) to alter the activityof the protein, including substitutions to one or more of the substratebinding site and/or active site. Such mutants can be informed by enzymestructures available in the art, including Yajima S, et al., Structureof 1-deoxy-D-xylulose 5-phosphate reductoisomerase in a quaternarycomplex with a magnesium ion, NADPH and the antimalarial drugfosmidomycin, Acta Cryst. F63, 466-470 (2007) and Perez-Gil, et al.,Crystal structure of brucella abortus deoxyxylulose-5-phosphatereductoisomerase-like (DRL) enzyme involved in isoprenoid biosynthesis,Journal of Biological Chemistry, 287(19): 15803-15809 (2012).

In some embodiments, wild-type IspE activity is complemented with one ormore additional gene copies, which may encode the wild-type enzyme, ormay encode a modified enzyme with one or more amino acid modifications(e.g., from one to ten modifications independently selected fromsubstitutions, insertions, and deletions) to increase enzyme activity orstability. In some embodiments, the bacterial strain is complementedwith an IspE ortholog having higher activity than the E. coli or nativebacterial enzyme. The E. coli IspE amino acid sequence is provided asSEQ ID NO: 7. In some embodiments, the bacterial strain expresses atleast one additional gene copy of ispE or a derivative, homolog, orortholog thereof. In some embodiments, the additional IspE enzyme is E.coli IspE or comprises one or more amino acid modifications (e.g., fromone to ten modifications independently selected from substitutions,insertions, and deletions) which may provide for increased activityand/or stability of the enzyme.

For example, in various embodiments the bacterial strain overexpressesor is complemented with an IspE enzyme having 50% or more sequenceidentity with SEQ ID NO: 7, or at least about 60% sequence identity, orat least about 70% sequence identity, or at least about 80% sequenceidentity, or at least about 90% sequence identity, or at least about 95%sequence identity, or at least 97% sequence identity with the amino acidsequence of SEQ ID NO: 7.

In some embodiments, the strain includes complementation with IspGand/or IspH. In some embodiments, the additional gene may be identicalor substantially identical to the native gene, or may be modified toincrease activity, or may be an IspG or IspH ortholog having higheractivity than the native bacterial (e.g., E. coli) enzyme. For example,with respect to IspG, the amino acid sequence may have 50% or moresequence identity with SEQ ID NO:5, or at least about 60% sequenceidentity, or at least about 70% sequence identity, or at least about 80%sequence identity, or at least about 90% sequence identity, or at leastabout 95% sequence identity, or at least about 97% sequence identitywith the amino acid sequence of SEQ ID NO:5. In some embodiments, from 1to about 10, or from 1 to about 5, amino acid substitutions, deletions,and/or insertions are made to the IspG amino acid sequence (SEQ ID NO:5)to alter the activity of the protein, including substitutions to one ormore of the substrate binding site or active site. Modifications to E.coli or other IspG can be informed by construction of a homology model.For example, a suitable homolog for construction of an E. coli IspGhomology model is disclosed in: Lee M, et al. Biosynthesis ofisoprenoids: crystal structure of the [4Fe-4S] cluster protein IspG. JMol Biol. 2010 Dec. 10; 404(4):600-10.

In some embodiments, the IspG enzyme contains one or more mutations atpositions selected from V30, S32, T34, N35, R37, V59, V61, S62, V63,L83, V84, C104, L105, P131, 1132, 1134, A138, K143, F176, K177, V178,V180, A182, L205, 1207, A210, G212, A213, L236, V238, A241, A242, D243,R259, 5262, R263, 1265, N266, F267, 1268, A269, T272, 5274, Q276, E277,F278, D289, 5301, 1302, 1303, V306. In some embodiments, modificationsare made at a plurality of positions selected from: (1) V30, S32, T34,N35, R37; or (2) V59, V61, S62, V63, L83, V84; or (3) C104, L105, 5301,1302, 1303, V306; or (4) P131, I132, I134, A138, K143; or (5) F176,K177, V178, V180, A182; or (6) L205, 1207, A210, G212, A213; or (7)L236, V238, A241, A242, D243; or (8) R259, S262, R263, 1265, N266; or(9) F267, 1268, A269, T272, S274, Q276, E277, F278, D289.

In some embodiments, the IspG enzyme has one, two, three or all of thefollowing mutations: L205V, A210S, G212T, and A213I.

Further, with respect to IspH, the amino acid sequence may have 50% ormore sequence identity with SEQ ID NO:6, or at least about 60% sequenceidentity, or at least about 70% sequence identity, or at least about 80%sequence identity, or at least about 90% sequence identity, or at leastabout 95% sequence identity, or at least 97% sequence identity with theamino acid sequence of SEQ ID NO:6. In some embodiments, from 1 to about10, or from 1 to about 5, amino acid substitutions, deletions, and/orinsertions are made to the IspH amino acid sequence (SEQ ID NO:6) toalter the activity of the protein, including substitutions to one ormore of the substrate binding site or active site. Modifications to theIspH enzyme can be informed by available IspH structures, includingGrawert, T., et al. Structure of active IspH enzyme from Escherichiacoli provides mechanistic insights into substrate reduction 2009 Angew.Chem. Int. Ed. Engl. 48: 5756-5759.

In these or other embodiments, pgi (glucose-6-phosphate isomerase)activity mutants (e.g., with reduced activity) are incorporated topotentially alter carbon flux, and may provide further improvements inproduct titer. In some embodiments, pgi is partially deleted orinactivated. In some embodiments, pgi contains from 1 to about 30 aminoacid substitutions, insertions, and/or deletions (e.g., about 1 to 20,or about 1 to 10 amino acid substitutions, insertions, and/or deletions)to alter or tune the activity of the enzyme for product titer or MEPcarbon.

The similarity of nucleotide and amino acid sequences, i.e. thepercentage of sequence identity, can be determined via sequencealignments. Such alignments can be carried out with several art-knownalgorithms, such as with the mathematical algorithm of Karlin andAltschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) orwith the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T.J. (1994) Nucleic Acids Res. 22, 4673-80). The grade of sequenceidentity (sequence matching) may be calculated using e.g. BLAST, BLAT orBlastZ (or BlastX). A similar algorithm is incorporated into the BLASTNand BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215: 403-410.BLAST polynucleotide searches can be performed with the BLASTN program,score=100, word length=12.

BLAST protein searches may be performed with the BLASTP program,score=50, word length=3. To obtain gapped alignments for comparativepurposes, Gapped BLAST is utilized as described in Altschul et al (1997)Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs are used.Sequence matching analysis may be supplemented by established homologymapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b,19 Suppl 1:154-162) or Markov random fields.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups:

(1) hydrophobic: Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro; and

(6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt α-helices. Some preferred conservativesubstitutions within the above six groups are exchanges within thefollowing sub-groups: (i) Ala, Val, Leu and Ile; (ii) Ser and Thr; (ii)Asn and Gin; (iv) Lys and Arg; and (v) Tyr and Phe.

As used herein, “non-conservative substitutions” are defined asexchanges of an amino acid by another amino acid listed in a differentgroup of the six standard amino acid groups (1) to (6) shown above.

Modifications of enzymes as described herein can include conservativeand/or non-conservative mutations.

In some embodiments “rational design” is involved in constructingspecific mutations in enzymes. Rational design refers to incorporatingknowledge of the enzyme, or related enzymes, such as its reactionthermodynamics and kinetics, its three dimensional structure, its activesite(s), its substrate(s) and/or the interaction between the enzyme andsubstrate, into the design of the specific mutation. Based on a rationaldesign approach, mutations can be created in an enzyme which can then bescreened for increased production of a terpene or terpenoid relative tocontrol levels. In some embodiments, mutations can be rationallydesigned based on homology modeling. As used herein, “homology modeling”refers to the process of constructing an atomic resolution model of oneprotein from its amino acid sequence and a three-dimensional structureof a related homologous protein.

In certain embodiments, the bacterial cell produces one or more terpeneor terpenoid compounds. A terpenoid, also referred to as an isoprenoid,is an organic chemical derived from a five-carbon isoprene unit (C5).Several non-limiting examples of terpenoids, classified based on thenumber of isoprene units that they contain, include: hemiterpenoids (1isoprene unit), monoterpenoids (2 isoprene units), sesquiterpenoids (3isoprene units), diterpenoids (4 isoprene units), sesterterpenoids (5isoprene units), triterpenoids (6 isoprene units), tetraterpenoids (8isoprene units), and polyterpenoids with a larger number of isopreneunits. In an embodiment, the bacterial host cell produces a terpenoidselected from a monoterpenoid, a sesquiterpenoid, diterpenoid, asesterpenoid, or a triterpenoid. Terpenoids represent a diverse class ofmolecules that provide numerous commercial applications, including inthe food and beverage industries as well as the perfume, cosmetic andhealth care industries. By way of example, terpenoid compounds find usein perfumery (e.g. patchoulol), in the flavor industry (e.g.,nootkatone), as sweeteners (e.g., steviol), or therapeutic agents (e.g.,taxol) and many are conventionally extracted from plants. Nevertheless,terpenoid molecules are found in ppm levels in nature, and thereforerequire massive harvesting to obtain sufficient amounts for commercialapplications.

The host cell will generally contain a recombinant downstream pathwaythat produces the terpenoid from IPP and DMAPP precursors. Terpenes suchas Monoterpenes (C10), Sesquiterpenes (C15), Diterpenes (C20),Sesterterpenes (C25), and Triterpenes (C30) are derived from the prenyldiphosphate substrates, geranyl diphosphate (GPP), farnesyl diphosphate(FPP), geranylgeranyl diphosphate (GGPP), geranylfarnesyl diphosphate(FGPP), and FPP, respectively, through the action of a very large groupof enzymes called the terpene (terpenoid) synthases. These enzymes areoften referred to as terpene cyclases since the product of the reactionsare cyclized to various monoterpene, sesquiterpene, diterpene,sesterterpene and triterpene carbon skeleton products. Many of theresulting carbon skeletons undergo subsequence oxygenation by cytochromeP450 enzymes to give rise to large families of derivatives.

Exemplary cytochrome P450 enzymes that are operative on diterpene andsesquiterpene scaffolds are described in WO 2016/073740 and WO2016/029153, which are hereby incorporated by reference. In addition,cytochrome P450 reductase proteins that find use in the bacterialstrains described herein are described in WO 2016/029153 as well as WO2016/073740.

The product of the invention in some embodiments is one or moreoxygenated terpenoids. As used herein, the term “oxygenated terpenoid”refers to a terpene scaffold having one or more oxygenation events,producing a corresponding alcohol, aldehyde, carboxylic acid and/orketone. In some embodiments, the bacterial cell produces at least oneterpenoid selected from Abietadiene, Abietic Acid, alpha-Sinensal,artemisinic acid, beta-Thuj one, Camphor, Carveol, Carvone, Celastrol,Ceroplastol, Cineole, Citral, Citronellal, Cubebol, Cucurbitane,Forskolin, Gascardic Acid, Geraniol, Haslene, Levopimaric Acid,Limonene, Lupeol, Menthol, Menthone, Mogroside, Nootkatone, Nootkatol,Ophiobolin A, Patchouli, Piperitone, Rebaudioside D (RebD), RebaudiosideM (RebM), Sabinene, Steviol, Steviol glycoside, Taxadiene, Thymol, andUrsolic Acid.

In some embodiments, the terpenoid synthase enzyme is upgraded toenhance the kinetics, stability, product profile, and/or temperaturetolerance of the enzyme, as disclosed, for example, in WO 2016/029153and WO 2016/073740, which are hereby incorporated by reference.

In another embodiment, the bacterial cell produces valencene and/ornootkatone. In such an embodiment, the bacterial cell may express abiosynthetic pathway that further includes a farnesyl diphosphatesynthase, a Valencene Synthase, and a Valencene Oxidase. Farnesyldiphosphate synthases (FPPS) produce farnesyl diphosphates fromiso-pentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Anexemplary farnesyl diphosphate synthase is ERG20 of Saccharomycescerevisiae (NCBI accession P08524) and E. coli ispA. Valencene synthaseproduces sesquiterpene scaffolds and are described in, for example, US2012/0107893, US 2012/0246767, and U.S. Pat. No. 7,273,735, which arehereby incorporated by reference in their entireties. Genes and hostcells for the production of terpenoid product comprising valenceneand/or nootkatone are described in WO 2016/029153, which is herebyincorporated by reference.

In an embodiment, the bacterial cell produces steviol or steviolglycoside (e.g., RebD or RebM). Steviol is produced from kaurene by theaction of two P450 enzymes, kaurene oxidase (KO) and kaurenoic acidhydroxylase (KAH). After production of steviol, various steviolglycoside products may be produced through a series of glycosylationreactions, which can take place in vitro or in vivo. Pathways andenzymes for production of steviol and steviol glycosides are disclosedin US 2013/0171328, US 2012/0107893, WO 2012/075030, WO 2014/122328,which are hereby incorporated by reference in their entireties. WO2016/073740 further discloses enzymes and bacterial host cells forproduction of RebM.

Other biosynthetic pathways for production of terpene or terpenoidcompounds are disclosed in U.S. Pat. No. 8,927,241, which is herebyincorporated by reference in its entirety.

The bacterial host cell is cultured to produce the terpenoid product,and with enhanced MEP pathway flux. In some embodiments, carbonsubstrates such as C1, C2, C3, C4, C5, and/or C6 carbon substrates areemployed for production of the terpene or terpenoid product. Inexemplary embodiments, the carbon source is glucose, sucrose, fructose,xylose, and/or glycerol. Culture conditions are generally selected fromaerobic, microaerobic, and anaerobic.

In various embodiments, the bacterial host cell may be cultured at atemperature between 22° C. and 37° C. While commercial biosynthesis inbacteria such as E. coli can be limited by the temperature at whichoverexpressed and/or foreign enzymes (e.g., enzymes derived from plants)are stable, recombinant enzymes (including the terpenoid synthase) maybe engineered to allow for cultures to be maintained at highertemperatures, resulting in higher yields and higher overallproductivity. In some embodiments, the culturing is conducted at about22° C. or greater, about 23° C. or greater, about 24° C. or greater,about 25° C. or greater, about 26° C. or greater, about 27° C. orgreater, about 28° C. or greater, about 29° C. or greater, about 30° C.or greater, about 31° C. or greater, about 32° C. or greater, about 33°C. or greater, about 34° C. or greater, about 35° C. or greater, about36° C. or greater, or about 37° C.

In some embodiments, the bacterial host cells are further suitable forcommercial production, at commercial scale. In some embodiments, thesize of the culture is at least about 100 L, at least about 200 L, atleast about 500 L, at least about 1,000 L, or at least about 10,000 L.In an embodiment, the culturing may be conducted in batch culture,continuous culture, or semi-continuous culture.

In various embodiments, methods further include recovering the terpeneor terpenoid product from the cell culture or from cell lysates. In someembodiments, the culture produces at least about 100 mg/L, or at leastabout 200 mg/L, or at least about 500 mg/L, or at least about 1 g/L, orat least about 2 g/L, or at least about 5 g/L, or at least about 10 g/L,or at least about 20 g/L, or at least about 30 g/L, or at least about 40g/L of the terpene or terpenoid product.

In some embodiments, the production of indole (including prenylatedindole) is used as a surrogate marker for terpenoid production, and/orthe accumulation of indole in the culture is controlled to increaseproduction. For example, in various embodiments, accumulation of indolein the culture is controlled to below about 100 mg/L, or below about 75mg/L, or below about 50 mg/L, or below about 25 mg/L, or below about 10mg/L. The accumulation of indole can be controlled by balancing proteinexpression and activity using the multivariate modular approach asdescribed in U.S. Pat. No. 8,927,241 (which is hereby incorporated byreference), and/or is controlled by chemical means.

Other markers for efficient production of terpene and terpenoids,include accumulation of DOX or ME in the culture media. Generally, thebacterial strains described herein accumulate less of these chemicalspecies, which accumulate in the culture at less than about 5 g/L, orless than about 4 g/L, or less than about 3 g/L, or less than about 2g/L, or less than about 1 g/L, or less than about 500 mg/L, or less thanabout 100 mg/L.

The optimization of terpene or terpenoid production by manipulation ofMEP pathway genes, as well as manipulation of the upstream anddownstream pathways, is not expected to be a simple linear or additiveprocess. Rather, through combinatorial analysis, optimization isachieved through balancing components of the MEP pathway, as well asupstream and downstream pathways. Indole (including prenylated indole)accumulation and MEP metabolite accumulation (e.g., DOX, ME, MEcPP,and/or farnesol) in the culture can be used as surrogate markers toguide this process.

For example, in some embodiments, the bacterial strain has at least oneadditional copy of dxs and idi expressed as an operon/module; or dxs,ispD, ispF, and idi expressed as an operon or module (either on aplasmid or integrated into the genome), with additional MEP pathwaycomplementation described herein to improve MEP carbon. For example, thebacterial strain may have a further copy of dxr, and ispG and/or ispH,optionally with a further copy of ispE and/or idi, with expressions ofthese genes tuned to increase MEP carbon and/or improve terpene orterpenoid titer. In various embodiments, the bacterial strain has afurther copy of at least dxr, ispE, ispG and ispH, optionally with afurther copy of idi, with expressions of these genes tuned to increaseMEP carbon and/or improve terpene or terpenoid titer

Manipulation of the expression of genes and/or proteins, including genemodules, can be achieved through various methods. For example,expression of the genes or operons can be regulated through selection ofpromoters, such as inducible or constitutive promoters, with differentstrengths (e.g., strong, intermediate, or weak). Several non-limitingexamples of promoters of different strengths include Trc, T5 and T7.Additionally, expression of genes or operons can be regulated throughmanipulation of the copy number of the gene or operon in the cell. Insome embodiments, expression of genes or operons can be regulatedthrough manipulating the order of the genes within a module, where thegenes transcribed first are generally expressed at a higher level. Insome embodiments, expression of genes or operons is regulated throughintegration of one or more genes or operons into the chromosome.

Optimization of protein expression can also be achieved throughselection of appropriate promoters and ribosomal binding sites. In someembodiments, this may include the selection of high-copy numberplasmids, or single-, low- or medium-copy number plasmids. The step oftranscription termination can also be targeted for regulation of geneexpression, through the introduction or elimination of structures suchas stem-loops.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA. The heterologous DNA is placed under operable controlof transcriptional elements to permit the expression of the heterologousDNA in the host cell.

In some embodiments, endogenous genes are edited, as opposed to genecomplementation. Editing can modify endogenous promoters, ribosomalbinding sequences, or other expression control sequences, and/or in someembodiments modifies trans-acting and/or cis-acting factors in generegulation. Genome editing can take place using CRISPR/Cas genomeediting techniques, or similar techniques employing zinc fingernucleases and TALENs. In some embodiments, the endogenous genes arereplaced by homologous recombination.

In some embodiments, genes are overexpressed at least in part bycontrolling gene copy number. While gene copy number can be convenientlycontrolled using plasmids with varying copy number, gene duplication andchromosomal integration can also be employed. For example, a process forgenetically stable tandem gene duplication is described in US2011/0236927, which is hereby incorporated by reference in its entirety.

The terpene or terpenoid product can be recovered by any suitableprocess, including partitioning the desired product into an organicphase or hydrophobic phase. Alternatively, the aqueous phase can berecovered, and/or the whole cell biomass can be recovered, for furtherprocessing. The production of the desired product can be determinedand/or quantified, for example, by gas chromatography (e.g., GC-MS). Thedesired product can be produced in batch or continuous bioreactorsystems. Production of product, recovery, and/or analysis of the productcan be done as described in US 2012/0246767, which is herebyincorporated by reference in its entirety. For example, in someembodiments, product oil is extracted from aqueous reaction medium usingan organic solvent, such as an alkane such as heptane or dodecane,followed by fractional distillation. In other embodiments, product oilis extracted from aqueous reaction medium using a hydrophobic phase,such as a vegetable oil, followed by organic solvent extraction andfractional distillation. Terpene and terpenoid components of fractionsmay be measured quantitatively by GC/MS, followed by blending offractions to generate a desired product profile.

In various embodiments, the recovered terpene or terpenoid isincorporated into a product (e.g., a consumer or industrial product).For example, the product may be a flavor product, a fragrance product, asweetener, a cosmetic, a cleaning product, a detergent or soap, or apest control product. For example, in some embodiments, the productrecovered comprises nootkatone, and the product is a flavor productselected from a beverage, a chewing gum, a candy, or a flavor additive,or the product is an insect repellant or insecticide. In someembodiments, the oxygenated product is steviol or a steviol glycoside(e.g., RebM), which is provided as a sweetener, or is incorporated intoingredients, flavors, beverages or food products.

The invention further provides methods of making products such as foods,beverages, texturants (e.g., starches, fibers, gums, fats and fatmimetics, and emulsifiers), pharmaceutical products, tobacco products,nutraceutical products, oral hygiene products, and cosmetic products, byincorporating the terpene or terpenoids produced herein. The higheryields of such species produced in embodiments of the invention canprovide significant cost advantages as well as sustainability.

In other aspects, the invention provides bacterial cells, such as E.coli, having one or more genetic modifications that increase MEP carbon.In various embodiments, the bacterial cells produce isopentenyldiphosphate (IPP) and dimethylallyl diphosphate (DMAPP) through the MEPpathway, and convert the IPP and DMAPP to a terpene or terpenoid productthrough a downstream synthesis pathway. The downstream synthesis pathwayis generally a recombinant pathway, and may comprise a prenyltransferase, a terpene synthase, and optionally one or more cytochromeP450 enzymes and cytochrome P450 reductase enzymes (for example, each asdescribed above). Further, to improve MEP carbon, the E. coli has one ormore of the following genetic modifications:

(1) decreased expression or activity of IspB, optionally by modificationof the RBS, and optionally where the six-base SD sequence in the ispBgene is changed to CGTGCT or CGTGCC, or a modification thereof havingone or two nucleotide changes from CGTGCT or CGTGCC;

(2) a deletion or inactivation of all or part of the iscR gene, and aconstitutive or inducible promoter to drive expression of the genesremaining in the isc operon (e.g., iscSUA) under conditions used forterpene or terpenoid production;

(3) a ryhB deletion or inactivation;

(4) MEP enzyme expression or activity is altered or balanced such thatDOX and/or ME do not accumulate above about 2 g/L or about 1 g/L;

(5) MEP pathway complementation comprising a dxr gene (optionally withispD, ispE, and/or ispF) and an ispG and/or ispH gene, where the IspG orIspH is optionally modified to increase enzyme activity; and wherein theIspG optionally has one or more mutations at positions selected fromV30, S32, T34, N35, R37, V59, V61, S62, V63, L83, V84, C104, L105, P131,1132, 1134, A138, K143, F176, K177, V178, V180, A182, L205, I207, A210,G212, A213, L236, V238, A241, A242, D243, R259, 5262, R263, 1265, N266,F267, 1268, A269, T272, 5274, Q276, E277, F278, D289, 5301, 1302, 1303,V306; and optionally modifications at a plurality of positions selectedfrom: (1) V30, S32, T34, N35, R37; or (2) V59, V61, S62, V63, L83, V84;or (3) C104, L105, 5301, 1302, 1303, V306; or (4) P131, 1132, 1134,A138, K143; or (5) F176, K177, V178, V180, A182; or (6) L205, 1207,A210, G212, A213; or (7) L236, V238, A241, A242, D243; or (8) R259,S262, R263, 1265, N266; or (9) F267, 1268, A269, T272, S274, Q276, E277,F278, D289; and optionally one, two, three, or all of L205V, A210S,G212T, and A213I; and

(6) a mutation in pgi (glucose-6-phosphate isomerase) supportingincreased terpenoid titer or increases in MEP carbon.

In some embodiments, the bacterial strain has at least one additionalcopy of dxs and idi expressed as an operon/module; or dxs, ispD, ispF,and idi expressed as an operon or module (either on a plasmid orintegrated into the genome). Further, the bacterial strain may have afurther copy of dxr, ispE, and ispG and/or ispH, with expressions ofthese genes tuned to increase MEP carbon and/or improve terpene orterpenoid titer.

In various embodiments, the bacterial strains comprises at least 2, atleast 3, at least 4, at least 5, or all 6 modifications defined by (1)to (6) above.

Genes can be overexpressed by complementation with recombinant genes, orthe endogenous genes can be modified to alter expression, as disclosedelsewhere herein.

The bacterial strain is a bacteria selected from Escherichia spp.,Bacillus spp., Corynebacterium spp., Rhodobacter spp., Zymomonas spp.,Vibrio spp., and Pseudomonas spp. For example, the bacterial strain is aspecies selected from Escherichia coli, Bacillus subtilis,Corynebacterium glutamicum, Rhodobacter capsulatus, Rhodobactersphaeroides, Zymomonas mobilis, Vibrio natriegens, or Pseudomonasputida. In some embodiments, the bacterial strain is E. coli.

Aspects and embodiments of the invention are further demonstrated belowwith reference to the following Examples.

EXAMPLES Example 1: Increased Availability of Fe—S Proteins ShiftsAccumulation from DOX to ME

As shown in FIG. 3, G2 (as described in FIG. 2) produces keyextracellular metabolites (DOX+ME+MEcPP) equivalent to 3.45 g/L ofterpenoid product. This compares to 7 g/L of primarily DOX in the strainnot containing modifications to increase Fe—S proteins. G4 also had a56% higher accumulation of the terpenoid product. Genetic modificationsinclude AryhB, AiscR with overexpression of the isc operon.

Similar results were obtained for an E. coli strain engineered toproduce another terpenoid product (Product B), and showing DOX+ME+MEcPPequivalent to 3.26 g/L of the terpenoid product. Additionalmodifications include a modification to pgi, which lead to a 1.4×improvement, and tuning of ispB translation. See FIG. 4.

As shown in FIG. 5, transcriptome sequencing and analysis comparingengineered product strains to wild-type E. coli generated strongevidence for involvement of ryhB and the isc operon in the alteredphenotypes of the product strains. The former gene is stronglyupregulated in our engineered strains, while the iscR regulatory gene isvery strongly down-regulated.

As shown in FIG. 6, sequential modification via deletion of ryhB, switchto constitutive overexpression for iscSUA, and deletion of iscR resultsin strains with steadily increasing product titers.

Example 2: Engineering ispG to Improve MEP Pathway Flux

A series of libraries where the active site of ispG was combinatoriallyvaried were created and tested in vivo by replacing the wild-type ispGgene with mutated versions. ispG engineering may improve kinetics,stability, and robustness of the enzyme, to relieve the impact of Fe—Scluster biochemistry on the flux through the MEP pathway. Nine activesite combinatorial libraries were designed to target substrate andiron-sulfur cluster binding sites using sequence alignments of ˜1000diverse ispG orthologs (FIG. 7A, 7B).

FIG. 8 shows the results of a primary screen of ispG combinatoriallibrary for increased sesquiterpene product titer in two backgroundstrains. Screening is conducted in 96 DWP at 37° C. for 48 hours.Similar performance was seen using both strain backgrounds (with orwithout ispH overexpression) with a total of ˜30 variants showing 1.2 to1.45× improvements in terpenoid product titers.

Lead variants were rescreened, with the results shown in FIG. 9. TwoispG variants gave ˜1.2× improvement in terpene product titer over thestrain engineered for increased availability of Fe—S proteins. The leadvariant, G11, has the following mutations: L205V, A210S, G212T, andA213I.

The lead variant is integrated into lead terpenoid producing strains,and the product titers shown in FIG. 10. Integration of ispG Lib6 G11led to a 1.2× improvement in production in both strains.

Example 3: ispB Translation Engineering

The ispB gene encodes an octaprenyl diphosphate synthase that controlsthe synthesis of ubiquinone and thus directly competes with FPP synthasefor IPP and DMAPP precursors. Decreasing the amount of ispB enzyme inthe cell by modifying the ispB RBS sequence and tuning down translationof the mRNA could shift carbon flux toward the recombinant terpenoidpathway.

As shown in FIG. 11, several potential hits are identified from primaryscreening of the ispB RBS libraries yielding up to 1.7 fold improvementin terpene/terpenoid production. Lead hits were validated as shown inFIG. 12. Two unique hits were identified that yielded a 1.5-foldimprovement in terpenoid production, having the following SD sequences:CGTGCT and CGTGCC.

Example 4: MEP Pathway Complementation

As shown in FIG. 13, overexpression of dxr by itself or in combinationwith ispD, ispE, and ispF decreases production by up to 2-fold. Additionof ispG-ispH returns production back to original levels.

As shown in FIG. 14, metabolomics analysis shows that overexpression ofdxr without ispE leads to accumulation of extracellular ME andintracellular CDP-ME. Overexpressing ispE shifts metabolite pools fromME and CDP-ME to MEcPP. In this example, strains are grown in 96-wellplates at 37 dC for 48 hrs. At the endpoint, the cell cultures aremeasured for OD600, then are split into two samples; the first isextracted with Ethyl Acetate to analyze and quantify Product A, whilethe second is centrifuged to pellet the cells, and the supernatant isanalyzed for extracellular MEP metabolites while the cell pellet isfurther processed to extract intracellular MEP metabolites. Theterpenoid product-containing organic phase sample is analyzed via gaschromatography and mass spectrometry (GC/MS), while the extracellularand intracellular metabolite samples are separated and analyzed vialiquid chromatography and mass spectrometry (LC/MS). The LC/MS detectoris a triple-quadrupole instrument, enabling accurate quantification ofMEP metabolites against authentic standards. The resulting metaboliteconcentrations are expressed in terms of molarity, to focus on the flowof carbon molecules through the MEP pathway to the desired product.

As shown in FIG. 15, metabolomics analysis of Product A G5 strains withMEP complementation with dxr or dxr-ispE further shows that theincreased MEP flux potential in the resulting strains translates intomore potential Product A titer. The measured product and MEP metaboliteconcentrations for these strain cultures are converted to molarity, andthe carbon equivalent of each MEP metabolite in terms of Product A iscalculated; i.e., one MEcPP molecule contains 5 carbons, so each onetranslates into 1 molecule of isopentenyl diphosphate (IPP, with 5carbons as well), or ⅓ of a sesquiterpene Product A molecule (with 15carbons). In this way, the potential total of Product A produced by thisstrain—when carbon is successfully pulled downstream through MEcPP—canbe calculated. With balanced expression between MEP genes, up to 12×more product A could result.

LISTING OF SEQUENCES SEQ ID NO: 1 (modified Shine-Dalgarno sequence 1):CGTGCT SEQ ID NO: 2 (modified Shine-Dalgarno sequence 2): CGTGCCSEQ ID NO: 3 (E. coli IspB)MNLEKINELTAQDMAGVNAAILEQLNSDVQLINQLGYYIVSGGGKRIRPMIAVLAARAVGYEGNAHVTIAALIEFIHTATLLHDDVVDESDMRRGKATANAAFGNAASVLVGDFIYTRAFQMMTSLGSLKVLEVMSEAVNVIAEGEVLQLMNVNDPDITEENYMRVIYSKTARLFEAAAQCSGILAGCTPEEEKGLQDYGRYLGTAFQLIDDLLDYNADGEQLGKNVGDDLNEGKPTLPLLHAMHHGTPEQAQMIRTAIEQGNGRHLLEPVLEAMNACGSLEWTRQRAEEEADKAIAALQVLPDTPWREALIGLAHIAVQRDR SEQ ID NO: 4 (E. coli Dxr)MKQLTILGSTGSIGCSTLDVVRHNPEHFRVVALVAGKNVTRMVEQCLEFSPRYAVMDDEASAKLLKTMLQQQGSRTEVLSGQQAACDMAALEDVDQVMAAIVGAAGLLPTLAAIRAGKTILLANKESLVTCGRLFMDAVKQSKAQLLPVDSEHNAIFQSLPQPIQHNLGYADLEQNGVVSILLTGSGGPFRETPLRDLATMTPDQACRHPNWSMGRKISVDSATMMNKGLEYIEARWLFNASASQMEVLIHPQSVIHSMVRYQDGSVLAQLGEPDMRTPIAHTMAWPNRVNSGVKPLDFCKLSALTFAAPDYDRYPCLKLAMEAFEQGQAATTALNAANEITVAAFLAQQIRFTDIAALNLSVLEKMDMREPQCVDDVLSVDANAREVARKE VMRLASSEQ ID NO: 5 (E. coli IspG)MHNQAPIQRRKSTRIYVGNVPIGDGAPIAVQSMTNTRTTDVEATVNQIKALERVGADIVRVSVPTMDAAEAFKLIKQQVNVPLVADIHFDYRIALKVAEYGVDCLRINPGNIGNEERIRMVVDCARDKNIPIRIGVNAGSLEKDLQEKYGEPTPQALLESAMRHVDHLDRLNFDQFKVSVKASDVFLAVESYRLLAKQIDQPLHLGITEAGGARSGAVKSAIGLGLLLSEGIGDTLRVSLAADPVEEIKVGFDILKSLRIRSRGINFIACPTCSRQEFDVIGTVNALEQRLEDIITPMDVSIIGCVVNGPGEALVSTLGVTGGNKKSGLYEDGVRKDRLDNNDMIDQLEARIRAKASQLDEARRIDVQQVEK SEQ ID NO: 6 (E. coli IspH)MQILLANPRGFCAGVDRAISIVENALAIYGAPIYVRHEVVHNRYVVDSLRERGAIFIEQISEVPDGAILIFSAHGVSQAVRNEAKSRDLTVFDATCPLVTKVHMEVARASRRGEESILIGHAGHPEVEGTMGQYSNPEGGMYLVESPDDVWKLTVKNEEKLSFMTQTTLSVDDTSDVIDALRKRFPKIVGPRKDDICYATTNRQEAVRALAEQAEVVLVVGSKNSSNSNRLAELAQRMGKRAFLIDDAKDIQEEWVKEVKCVGVTAGASAPDILVQNVVARLQQLGGGEAIPLE GREENIVFEVPKELRVDIREVDSEQ ID NO: 7 (Brucella abortus Deoxyxylulose-5-phosphate Reductoisom erase-like (DRL))MTTNVALVGLARDLAARAETGKPIRIGLIGAGEMGTDIVTQVARMQGIEVGALSARRLPNTFKAIRTAYGDEENAREATTESAMTRAIEAGKIAVTDDNDLILSNPLIDVIIDATGIPEVGAETGIAAIRNGKHLVMMNVEADVTIGPYLKAQADKQGVIYSLGAGDEPSSCMELIEFVSALGYEVVSAGKGKNNPLNFDATPDDYRQEADRRNMNVRLLVEFIDGSKTMVEMAAIANATGLVPDIAGMHGPRASIDQLSHTLIPQAEGGVLSKSGVVDYSIGKGVSPGVFVVAKMDHPRLNERLEDLKIGKGPYFTFHRPYHLTSLEVPLTVARVVLHGKTDMVPLPKPVAEVCAVAKKDMQPGEHLDAIGQYCYRSWIMTVPEARAAKAIPCGLLQNGTVIAPIKKGELITYANAAPQPGSRIAELRALQDAMLGQSEQ ID NO: 8 (E. coli ispE)MRTQWPSPAKLNLFLYITGQRADGYHTLQTLFQFLDYGDTISIELRDDGDIRLLTPVEGVEHEDNLIVRAARLLMKTAADSGRLPTGSGANISIDKRLPMGGGLGGGSSNAATVLVALNHLWQCGLSMDELAEMGLTLGADVPVFVRGHAAFAEGVGEILTPVDPPEKWYLVAHPGVSIPTPVIFKDPELPRNTPKRSIETLLKCEFSNDCEVIARKRFREVDAVLSWLLEYAPSRLTGTGACVFAEFDTESEARQVLEQAPEWLNGFVAKGANLSPLHRAML

1. A method for production of a terpene or terpenoid product,comprising: providing a bacterial strain that produces isopentenyldiphosphate (IPP) and dimethylallyl diphosphate (DMAPP) through anupstream methylerythritol pathway (MEP) and converts the IPP and DMAPPto a terpene or terpenoid product through a downstream synthesispathway; and culturing the bacterial strain to produce the terpene orterpenoid product, wherein greater than 15% of carbon enteringglycolysis becomes MEP carbon. 2.-6. (canceled)
 7. The method of claim1, wherein the ubiquinone biosynthesis pathway is downregulated.
 8. Themethod of claim 7, wherein the strain has reduced expression or activityof IspB.
 9. The method of claim 8, wherein IspB expression is reduced bya modified ribosome binding sequence (RBS).
 10. The method of claim 8,wherein the Shine-Dalgarno (SD) sequence of the ispB gene is CGTGCT orCGTGCC or a modification thereof having one or two nucleotide changes inCGTGCT or CGTGCC. 11.-13. (canceled)
 14. The method of claim 1, whereinthe strain has constitutive expression of the isc operon.
 15. The methodof claim 14, wherein the iscR gene is deleted in whole or in part, or isinactivated, optionally by modification to the RBS.
 16. The method ofclaim 14, wherein the iscR gene is replaced with a inducible orconstitutive promoter.
 17. The method of claim 1, wherein the straincomprises a ryhB deletion or inactivation.
 18. The method of claim 1,wherein the strain overexpresses Dxr, optionally by complementation witha recombinant gene or operon comprising dxr.
 19. The method of claim 18,wherein the strain has a modified Dxr enzyme or Dxr ortholog havingincreased activity with respect to the wild type E. coli Dxr.
 20. Themethod of claim 18, wherein the strain overexpresses IspE, a modifiedIspE, and/or an IspE ortholog, optionally by complementation with arecombinant gene or operon comprising ispE.
 21. The method of claim 18,wherein the strain overexpresses ispG and ispH, optionally bycomplementation with a recombinant gene or operon comprising ispG and H.22. The method of claim 21, wherein the strain has a modified IspGenzyme or ortholog having increased activity with respect to the wildtype E. coli IspG.
 23. The method of claim 22, wherein the IspG enzymecontains one or more mutations at positions selected from V30, S32, T34,N35, R37, V59, V61, S62, V63, L83, V84, C104, L105, P131, 1132, 1134,A138, K143, F176, K177, V178, V180, A182, L205, 1207, A210, G212, A213,L236, V238, A241, A242, D243, R259, 5262, R263, 1265, N266, F267, 1268,A269, T272, 5274, Q276, E277, F278, D289, 5301, 1302, 1303, V306. 24.The method of claim 23, wherein the IspG has a modification at aplurality of positions selected from: (1) V30, S32, T34, N35, R37; or(2) V59, V61, S62, V63, L83, V84; or (3) C104, L105, S301, I302, I303,V306; or (4) P131, I132, I134, A138, K143; or (5) F176, K177, V178,V180, A182; or (6) L205, 1207, A210, G212, A213; or (7) L236, V238,A241, A242, D243; or (8) R259, S262, R263, 1265, N266; or (9) F267,1268, A269, T272, S274, Q276, E277, F278, D289.
 25. The method of claim23, wherein the IspG enzyme has one, two, three or all of the followingmutations: L205V, A210S, G212T, and A213I.
 26. (canceled)
 27. The methodof claim 1, wherein the bacterial strain has at least one additionalcopy of dxs and idi expressed as an operon/module; or dxs, ispD, ispF,and idi expressed as an operon or module.
 28. The method of claim 1,wherein the strain comprises a pgi mutation supporting increased terpeneor terpenoid titer or increased MEP carbon.
 29. (canceled)
 30. Themethod of claim 1, wherein the strain is cultured with a C1, C2, C3, C4,C5, and/or C6 carbon source under microaerobic conditions. 31.(canceled)
 32. The method of claim 30, wherein the culture is at leastabout 100 L.
 33. (canceled)
 34. The method of claim 32, comprising,monitoring the accumulation of DOX, ME, or MEcPP in the culture.
 35. Themethod of claim 1, further comprising, recovering the terpene orterpenoid product.
 36. (canceled)
 37. A method for making an industrialor consumer product, comprising, incorporating the terpene or terpenoidmade according to the method of claim 1 into said industrial or consumerproduct. 38.-39. (canceled)
 40. The method of claim 19, wherein thestrain overexpresses DRL, a modified DRL, and/or an DRL ortholog,optionally by complementation with a recombinant gene or operoncomprising DRL. 41-52. (canceled)
 53. The method of claim 1, wherein thebacterial strain has a gene complementation or overexpression of one ormore MEP pathway genes, and DOX, ME, and MEcPP metabolites are found atreduced levels in the culture medium as compared to a parent strain. 54.The method of claim 53, wherein the bacterial strain has a genecomplementation of dxs, ispD, ispF, and idi genes.
 55. The method ofclaim 54, wherein the bacterial strain has an overexpression of ispG andispH.
 56. The method of claim 55, wherein the bacterial strain has agene complementation of dxr.
 57. The method of claim 56, wherein thebacterial strain has a gene complementation of ispE.
 58. The method ofclaim 53, wherein DOX or ME each accumulate in culture media at lessthan 3 g/L.
 59. The method of claim 53, wherein DOX or ME eachaccumulate in culture media at less than 2 g/L.
 60. The method of claim53, wherein DOX or ME each accumulate in culture media at less than 1g/L.